An elevated level of homocysteine in the blood appears to be an important indicator for many human disease states. Homocysteine is predictive of vascular disease and stroke, Ueland, P. M. (1992) and Kluijtmans L. A. J. et al (1996); is correlated with forms of diabetes and alcoholism, Cravo, M. L. et al (1996); is used to monitor liver and kidney damage, Bostom, A. G. et al (1996) and neural tube defects, Steegers-Theunissen, R. P. N. (1992) and is associated with certain inborn errors of metabolism, Mudd, S. H., (1989).
Homocysteine levels in blood are conventionally determined using high performance liquid chromatography (HPLC) methods, see for example at Poele-Pothoff M. T. B. et al, (1995). However, HPLC methods employ expensive and elaborate machinery, are generally sophisticated and are considered impractical for many routine analyses.
Patent publication WO93/15220 (Cockbain) describes a method for assaying homocysteine in blood using a homocysteine converting enzyme, S-adenosyl homocysteine hydrolase (SAH-hydrolase). SAH-hydrolase catalyses the conversion of homocysteine with a co-substrate, adenosine, to S-adenosyl-homocysteine. It is then possible, by determining the amount of adenosine consumed, to make a correlation with the amount of homocysteine consumed. The amount of homocysteine in a sample is then determined from differences in adenosine concentration. However, such an assay requires the use of two initial substrates (homocysteine and adenosine) and two enzymes, making it relatively complex. It also involves determining a decrease in the concentration of adenosine, which may not be satisfactory.
U.S. Pat. No. 4,940,658 (Allen et al) describes a method for determining sulphydryl amino acids, including homocysteine levels, in samples of body tissues, method of detecting cobalamin and folic acid deficiency using an assay for total homocysteine levels, and methods for distinguishing cobalamin from folic acid deficiency using an assay for total homocysteine levels in conjunction with an assay for methylmalonic acid. The assays comprise combining a sample with a reference standard comprising a known amount of a sulphydryl amino acid to be assayed, labelled with a suitable marker and measuring the relative amounts of labelled and unlabelled sulphydryl amino acid present for each species with a mass spectrometer. As the amount of labelled species is known, it is therefore possible from calculating the ratio of labelled to unlabelled species to determine the amount of sulphydryl amino acid present in the sample.
U.S. Pat. No. 5,438,017 describes a gas chromatography/mass spectrometry method for analysis of sulphydryl amino acids in a sample of body fluid. The assay relies on the use of a labelled reference sulphydryl amino acid, similar to that described in U.S. Pat. No. 4,940,658, but has additional treatment and/or purification steps prior to analysing the sample by gas chromatography/mass spectrometry.
It will be appreciated that similar to HPLC methods, the assays described above which employ gas chromatography/mass spectrometry are generally sophisticated, use expensive and elaborate machinery and are considered impractical for many routine analyses.